Postural Balance; Hearing; Neurobiology; Neurology; Neurosciences
Neurology: Navaratnam Lab
We work on the molecular basis of a number of physiological phenomena related to the hearing and balance organs.
Phenomenon of electrical resonance.
Electrical tuning is a phenomenon by which certain vertebrates discriminate between different frequencies of sound. Electrical resonance results when the inherent oscillation in the membrane potential of hair cells corresponds to sound of a particular frequency. This gives rise to a resonance and amplification of signal with consequent transmitter release from these cells. The inherent oscillation in membrane potential in a hair cell is brought about by an inward Calcium current and an outward Potassium current (calcium dependent). Inherent to this view is that the two proteins are physically proximate.
We had previously erroneously believed that the range in BK channel currents was brought about by alternative splicing. We now hypothesize that this variation in current is brought about by association with other proteins. We have isolated several binding partners using the yeast two hybrid technique and are in the process of evaluating their ability to alter BK kinetics and bring about channel clustering and co-localization. The role of Prestin (in Collaboration with Dr. Joseph Santos-Sacchi). Prestin is a recently described protein in outer hair cells that is responsible for the sharp tuning seen in the hearing organ of mammals. It is critical for normal hearing. Knocking out of this protein results in the loss of hearing in mice.
Specialized Terms: Hearing and Balance Organs
Extensive Research Description
Phenomenon of electrical resonance
Electrical tuning is a phenomenon by which certain vertebrates discriminate between different frequencies of sound. Electrical resonance results when the inherent oscillation in the membrane potential of hair cells corresponds to sound of a particular frequency. This gives rise to a resonance and amplification of signal with consequent transmitter release from these cells.
The inherent oscillation in membrane potential in a hair cell is brought about by an inward Calcium current and an outward Potassium current (calcium dependent). The systematic variation in the frequency of such an oscillation in hair cells that occurs across the tonotopic axis is brought about primarily by a variation in the kinetic properties of the Potassium current. Previously we had shown that some of this variation in kinetic properties of this current was brought about by alternative splicing of this BK potassium channel. However, a large part of this variation in kinetics cannot be explained by alternative splicing alone.
Our present hypothesis is that this variation in current is brought about by association with other proteins. Towards this end we have made yeast two hybrid libraries from the cochlea organ and are screening these libraries for binding proteins using the purported intracellular domains of the BK channel as “bait”. In addition we have also demonstrated by fluorescence immunohistochemistry that the calcium channel and the potassium channel are in fact in close physical approximation in these hair cells.
There is considerable theoretical reasons to support this observation. We have preliminary data that these two proteins are physically linked to one another. We are presently attempting to duplicate these results using a more sensitive method of detection (tandem mass spec at the Keck facility). We are also seeking to identify proteins that might serve as scaffolding linking both these channels. The role of Prestin (in Collaboration with Dr Joseph Santos Saatchi, Prof in Otolaryngology see www.yaleearlab.org) Prestin is a recently described protein in outer hair cells that is responsible for the sharp tuning seen in the hearing organ of mammals.
It is critical for normal hearing. Knocking out of this protein results in the loss of hearing in mice. Prestin is a “motor” protein that gives rise to electromotility in outer hair cells. Its an unusual phenomenon in that motility in outer hair cells can be rapid, upto 20KHz, which is orders of magnitude faster than conventional motor proteins like myosin. We are attempting to determine the functional domains within this protein by mutagenesis. We have made over a dozen mutants of this protein and are presently evaluating how it affects the motor function of this protein.
Regeneration in the auditory epithelium
Hearing loss in the elderly population affects upto 60% of this age group. This hearing loss is largely brought about by a loss in hair cells in the auditory epithelium. In previous experiments we had demonstrated that increasing levels of the second messenger cyclic AMP results in a cascade of events giving rise to cell division and regeneration in hair cells.
These results have since been duplicated in mice. We are attempting to identify the molecular events that underlie such regeneration and we have undertaken a subtractive hybridization experiment to determine the identity of mRNA transcripts that are upregulated with increasing cAMP. We have identified several molecules.
One such molecule is Id 2 a protein that has a binding domain but lacks the activation domain of BHLH transcription factors. Id2 is thought to repress other BHLH transcription factors by preventing their dimerization. To determine the effects of Id2 on regeneration we have used antisense oligos to suppress the expression of this protein in cochleas treated with forskolin (which increases cAMP levels).
Our preliminary data suggests that suppressing Id2 results in a reduced number of cells entering the cell cycle (measured by incorporation of BrDU). We are presently attempting to further corroborate this finding and will undertake experiments to determine the binding partners of Id2.
Cholinergic synaptic function
Acetylcholinesterase in cholinergic synapses removes acetylcholine. Acetylcholine, the neurotransmitter in cholinergic neurons, is rapidly removed from cholinergic synapses by hydrolysis by the enzyme acetylcholinesterase.
Failure to remove acetylcholine in a timely fashion gives rise to a sustained depolarization of the postsynaptic membrane and a failure to respond to further neurotransmitter release. Thus the timely removal of acetylcholine brought about by the localization of acetylcholinesterase in these synapses is critical for normal neurotransmission to occur. We have identified two proteins that likely serve to localize the protein to these neuronal synapses. One of these proteins is related to the bacterial Cu transporter CUTA1.
The other is a novel protein which however has a proline rich domain that has been shown to be important in interactions between acetylcholinesterase and the colq protein that localizes the enzyme to the synapse in the neuromuscular junction. We have made several mutants of the latter protein and are presently attempting to relate the ability of these mutants to alter the localization of acetylcholineterase to synaptic membranes.
Hair cells--beyond the transducer.
Housley GD, Marcotti W, Navaratnam D, Yamoah EN. Hair cells--beyond the transducer. The Journal Of Membrane Biology 2006, 209:89-118.
En block C-terminal charge cluster reversals in prestin (SLC26A5): effects on voltage-dependent electromechanical activity.
Bai JP, Navaratnam D, Samaranayake H, Santos-Sacchi J. En block C-terminal charge cluster reversals in prestin (SLC26A5): effects on voltage-dependent electromechanical activity. Neuroscience Letters 2006, 404:270-5.
- Santos-Sacchi, J, Song, L, Bai, J-P, and Navaratnam, D. Chloride and the OHC lateral membrane motor. Mechanics of Hearing 2006 in the press
Developmental expression of the outer hair cell motor prestin in the mouse.
Abe T, Kakehata S, Kitani R, Maruya S, Navaratnam D, Santos-Sacchi J, Shinkawa H. Developmental expression of the outer hair cell motor prestin in the mouse. The Journal Of Membrane Biology 2007, 215:49-56.
Full List of PubMed Publications
- Fleming MR, Brown MR, Kronengold J, Zhang Y, Jenkins DP, Barcia G, Nabbout R, Bausch AE, Ruth P, Lukowski R, Navaratnam DS, Kaczmarek LK: Stimulation of Slack K(+) Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex. Cell Rep. 2016 Aug 30; 2016 Aug 18. PMID: 27545877
- Moeini-Naghani I, Navaratnam DS: Yeast Two-Hybrid Screening to Test for Protein-Protein Interactions in the Auditory System. Methods Mol Biol. 2016. PMID: 27259923
- Zhang Y, Moeini-Naghani I, Bai J, Santos-Sacchi J, Navaratnam DS: Tyrosine motifs are required for prestin basolateral membrane targeting. Biol Open. 2015 Jan 16; 2015 Jan 16. PMID: 25596279
- Frucht CS, Uduman M, Kleinstein SH, Santos-Sacchi J, Navaratnam DS: Gene expression gradients along the tonotopic axis of the chicken auditory epithelium. J Assoc Res Otolaryngol. 2011 Aug; 2011 Mar 12. PMID: 21399991
- Frucht CS, Santos-Sacchi J, Navaratnam DS: MicroRNA181a plays a key role in hair cell regeneration in the avian auditory epithelium. Neurosci Lett. 2011 Apr 8; 2011 Feb 21. PMID: 21316421
- Bian S, Bai JP, Chapin H, Le Moellic C, Dong H, Caplan M, Sigworth FJ, Navaratnam DS: Interactions between β-catenin and the HSlo potassium channel regulates HSlo surface expression. PLoS One. 2011; 2011 Dec 14. PMID: 22194818
- Bian S, Koo BW, Kelleher S, Santos-Sacchi J, Navaratnam DS: A highly expressing Tet-inducible cell line recapitulates in situ developmental changes in prestin's Boltzmann characteristics and reveals early maturational events. Am J Physiol Cell Physiol. 2010 Oct; 2010 Jul 14. PMID: 20631244
- Frucht CS, Uduman M, Duke JL, Kleinstein SH, Santos-Sacchi J, Navaratnam DS: Gene expression analysis of forskolin treated basilar papillae identifies microRNA181a as a mediator of proliferation. PLoS One. 2010 Jul 9; 2010 Jul 9. PMID: 20634979
- Navaratnam DS: Yeast two-hybrid screening to test for protein-protein interactions in the auditory system. Methods Mol Biol. 2009. PMID: 18839352
- Samaranayake H, Saunders JC, Greene MI, Navaratnam DS: Ca(2+) and K(+) (BK) channels in chick hair cells are clustered and colocalized with apical-basal and tonotopic gradients. J Physiol. 2004 Oct 1; 2004 Jul 22. PMID: 15272029